Mammalian cell surface antigens; related reagents

ABSTRACT

Purified genes encoding a T cell surface antigen from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding this antigen are provided. Methods of using said reagents and diagnostic kits are also provided.

This application is a Divisional of 10/326,052, filed Dec. 23, 2002,which is a Continuation of U.S. patent application Ser. No. 09/671,658,filed Sep. 27, 2000, now U.S. Pat. No. 6,525,180, which is aContinuation of U.S. patent application Ser. No. 08/989,362, filed Dec.12, 1997, now U.S. Pat. No. 6,242,586, which claims benefit of U.S.Provisional Patent Application No. 60/032,846, filed Dec. 13, 1996, eachof which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention pertains to compositions related to proteins whichfunction in controlling activation and expansion of mammalian cells,e.g., cells of a mammalian immune system. In particular, it providespurified genes, proteins, antibodies, and related reagents useful, e.g.,to regulate activation, development, differentiation, and function ofvarious cell types, including hematopoietic cells.

BACKGROUND OF THE INVENTION

The activation of resting T cells is critical to most immune responsesand allows these cells to exert their regulatory or effectorcapabilities. See Paul (ed; 1993) Fundamental Immunology 3d ed., RavenPress, N.Y. Increased adhesion between T cells and antigen presentingcells (APC) or other forms of primary stimuli, e.g., immobilizedmonoclonal antibodies (mAb), can potentiate the T-cell receptor signals.T-cell activation and T cell expansion depends upon engagement of theT-cell receptor (TCR) and co-stimulatory signals provided by accessorycells. See, e.g., Jenkins and Johnson (1993) Curr. Opin. Immunol.5:361-367; Bierer and Hahn (1993) Semin. Immunol. 5:249-261; June, etal. (1990) Immunol. Today 11:211-216; and Jenkins (1994) Immunity1:443-446. A major, and well-studied, co-stimulatory interaction for Tcells involves either CD28 or CTLA-4 on T cells with either B7 or B70(Jenkins (1994) Immunity 1:443-446). Recent studies on CD28 deficientmice (Shahinian, et al. (1993) Science 261:609-612; Green, et al. (1994)Immunity 1:501-508) and CTLA-4 immunoglobulin expressing transgenic mice(Ronchese, et al. (1994) J. Exp. Med. 179:809-817) have revealeddeficiencies in some T-cell responses though these mice have normalprimary immune responses and normal CTL responses to lymphocyticchoriomeningitis virus and vesicular stomatitis virus, As a result, boththese studies conclude that other co-stimulatory molecules must besupporting T-cell function. However, identification of these moleculeswhich mediate distinct costimulatory signals has been difficult.

Tumor Necrosis Factor (TNF) is the prototypic member of an emergingfamily of cytokines that function as prominent mediators of immuneregulation and the inflammatory response. These ligands are typicallytype II membrane proteins, with homology at the carboxy terminus. Aproteolytic processed soluble protein often is produced. See, e.g.,Smith, et al. (1994) Cell 76-959-962; Armitage (1994) Current Opinion inImmunology 6:407-413; Gruss and Dower (1995) Blood 85:3378-3404; Wiley,et al. (1995) Immunity 3:673-682; and Baker and Reddy (1996) Oncogene12:1-9. Crucial roles for these family members are evidenced by a numberof studies, and they are implicated in regulation of apoptosis,peripheral tolerance, Ig maturation and isotype switching, and general Bcell and T cell functions. See, e.g., Thomson (ed. 1994) The cytokineHandbook Academic Press, San Diego, Calif. These imply fundamental rolesin immune and developmental networks.

The inability to modulate activation signals prevents control ofinappropriate developmental or physiological responses in the immunesystem. The present invention provides at least one alternativecostimulatory molecule, agonists and antagonists of which will be usefulin modulating a plethora of immune responses.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery of anantigen which exhibits sequence homology to proteins which act asinducers of apoptosis. In particular, it provides a gene encoding a 316amino acid protein, designated 499E9, which is expressed on a highlypolarized Th1 T cell. Engagement of 499E9 may modulate antigen-specificproliferation and cytokine production by effector cells. 499E9 is anovel cell surface molecule which, when engaged, may either potentiateimmune cell expansion or apoptosis. The mouse embodiment is described,enabling mammalian genes, proteins, antibodies, and uses thereof.Functional equivalents exhibiting significant sequence homology areavailable from other mammalian, e.g., human, and non-mammalian species.Moreover, the receptor of 499E9 can function as its binding partner tostimulate other cells expressing the receptor.

More particularly, the present invention provides a composition ofmatter selected from: a substantially pure or recombinant 499E9 proteinor peptide exhibiting at least about 85% sequence identity over a lengthof at least about 12 amino acids to SEQ ID NO: 2; a natural sequence499E9 of SEQ ID NO: 2; or a fusion protein comprising 499E9 sequence.Certain embodiments include a substantially pure or isolated proteincomprising a segment exhibiting sequence identity to a correspondingportion of a 499E9, wherein: the homology is at least about 90% identityand the portion is at least about 9 amino acids; the homology is atleast about 80% identity and the portion is at least about 17 aminoacids; or the homology is at least about 70% identity and the portion isat least about 25 amino acids. Other embodiments include a compositionof matter, wherein the: 499E9 comprises a mature sequence of Table 1; orprotein or peptide: is from a warm blooded animal selected from amammal, including a rodent; comprises at least one polypeptide segmentof SEQ ID NO: 2; exhibits a plurality of portions exhibiting theidentity; is a natural allelic variant of 499E9; has a length at leastabout 30 amino acids; exhibits at least two non-overlapping epitopeswhich are specific for a mammalian 499E9; exhibits a sequence identityat least about 90% over a length of at least about 20 amino acids to arodent 499E9; exhibits at least two non-overlapping epitopes which arespecific for a rodent 499E9; exhibits a sequence identity at least about90% over a length of at least about 20 amino acids to a rodent 499E9; isglycosylated; is a synthetic polypeptide; is attached to a solidsubstrate; is conjugated to another chemical moiety; is a 5-fold or lesssubstitution from natural sequence; or is a deletion or insertionvariant from a natural sequence. Also provided are various compositions,e.g., comprising: a sterile 499E9 protein or peptide; or the 499E9protein or peptide and a carrier, wherein the carrier is: an aqueouscompound, including water, saline, and/or buffer; and/or formulated fororal, rectal, nasal, topical, or parenteral administration. Fusionproteins are provided, e.g., comprising: mature protein sequence ofTable 1; a detection or purification tag, including a FLAG, His6, or Igsequence; or sequence of another TNF-ligand protein. Kit embodiments areprovided, e.g., comprising such a protein or polypeptide, and: acompartment comprising the protein or polypeptide; and/or instructionsfor use or disposal of reagents in the kit.

Antibody, or binding compound embodiments include those comprising anantigen binding portion from an antibody, which specifically binds to anatural 499E9 protein, wherein: the protein is a rodent protein; thebinding compound is an Fv, Fab, or Fab2 fragment; the binding compoundis conjugated to another chemical moiety; or the antibody: is raisedagainst a peptide sequence of a mature polypeptide comprising sequenceof Table 1; is raised against a mature 499E9; is raised to a purified499E9; is immunoselected; is a polyclonal antibody; binds to a denatured499E9; exhibits a Kd to antigen of at least 30 μM; is attached to asolid substrate, including a bead or plastic membrane; is in a sterilecomposition; or is detectably labeled, including a radioactive orfluorescent label. Other embodiments include a kit comprising thebinding compound, and: a compartment comprising the binding compound;and/or instructions for use or disposal of reagents in the kit. Otherforms include, e.g., a composition comprising: a sterile bindingcompound; or the binding compound and a carrier, wherein the carrier is:an aqueous compound, including water, saline, and/or buffer; and/orformulated for oral, rectal, nasal, topical, or parenteraladministration. Such also allow methods of purifying a 499E9 protein orpeptide from other materials in a mixture comprising contacting themixture to such an antibody, and separating bound 499E9 from othermaterials.

Nucleic acid embodiments include an isolated or recombinant nucleic acidencoding a protein or peptide or fusion protein, wherein: the 499E9protein is from a mammal, including a rodent; or the nucleic acid:encodes an antigenic peptide sequence of Table 1; encodes a plurality ofantigenic peptide sequences of Table 1; exhibits at least about 80%identity to a natural cDNA encoding the segment; is an expressionvector; further comprises an origin of replication; is from a naturalsource; comprises a detectable label; comprises synthetic nucleotidesequence; is less than 6 kb, preferably less than 3 kb; is from amammal, including a rodent; comprises a natural full length codingsequence; is a hybridization probe for a gene encoding the 499E9protein; or is a PCR primer, PCR product, or mutagenesis primer. A cellor tissue comprising such a recombinant nucleic acid is also embracedwithin the invention, e.g., wherein the cell is: a prokaryotic cell; aeukaryotic cell; a bacterial cell; a yeast cell; an insect cell; amammalian cell; a mouse cell; a rodent cell; or a human cell. Kit formsinclude those comprising the nucleic acid, and: a compartment comprisingthe nucleic acid; a compartment further comprising a 499E9 protein orpolypeptide; and/or instructions for use or disposal of reagents in thekit. Other nucleic acid embodiments include those which: hybridize underwash conditions of: 30° C. and less than 2M salt, 45° C. and/or 500 mMsalt, or at 55° C. and/or 150 mM salt to SEQ ID NO: 1; or exhibit atleast about 85% identity over a stretch of at least about 30nucleotides, at least 90% and/or the stretch is at least 55 nucleotides,or at least 95% and/or the stretch is at least 75 nucleotides to arodent 499E9.

The invention further provides methods of modulating physiology ordevelopment of a cell or tissue culture cells comprising introducinginto the cell an agonist or antagonist of a 499E9. Other methods includemodulating the physiology of a cell comprising contacting the cell with:a substantially pure 499E9 or fragment; an antibody or binding partnerwhich specifically binds a 499E9; or c) a nucleic acid encoding a 499E9or peptide. Preferably, the is a T cell and the modulating of physiologyis: apoptosis of the T cell; or activation of the T cell.

The invention further provides a method of treating a patient having anabnormal immune response by administering an effective dose of anantibody or binding partner specific for 499E9; a 499E9 protein orpolypeptide; or a nucleic acid encoding a 499E9 peptide. The abnormalimmune response is characterized by a T cell immune deficiency; chronicinflammation; or tissue rejection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Outline

I. General

II. Purified 499E9

-   -   A. physical properties    -   B. biological properties

III. Physical Variants

-   -   A. sequence variants, fragments    -   B. post-translational variants        -   1. glycosylation        -   2. others

IV. Functional Variants

-   -   A. analogs, fragments        -   1. agonists        -   2. antagonists    -   B. mimetics        -   1. protein        -   2. chemicals    -   C. species variants

V. Antibodies

-   -   A. polyclonal    -   B. monoclonal    -   C. fragments, binding compositions

VI. Nucleic Acids

-   -   A. natural isolates; methods    -   B. synthetic genes    -   C. methods to isolate

VII. Making 499E9, mimetics

-   -   A. recombinant methods    -   B. synthetic methods    -   C. natural purification

VIII. Uses

-   -   A. diagnostic    -   B. therapeutic

IX. Kits

-   -   A. nucleic acid reagents    -   B. protein reagents    -   C. antibody reagents

X. Isolating a binding partner (ligand)

I. General

The present invention provides amino acid sequences and DNA sequencesencoding various mammalian proteins which are antigens found in many Tcell subtypes, e.g., Th1, Th2, polarized Th1 cells, and polarized Th2cells. Among these proteins are antigens which modulate, e.g., induce orprevent proliferation or differentiation of interacting cells, amongother physiological effects. The full length antigens, and fragments, orantagonists will be useful in physiological modulation of cellsexpressing counter receptors for the antigen. The proteins will also beuseful as antigens, e.g., immunogens, for raising antibodies to variousepitopes on the protein, both linear and conformational epitopes. Themolecule may be useful in defining functional T cell or NK cell subsets.

A cDNA encoding 499E9 was isolated from a polarized Th1 cell cDNAlibrary, see Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367. The499E9 cDNA contains a stretch of about 2191 bp in length and containedone large open reading frame encoding a type II transmembrane protein.Transcript analysis has identified multiple transcripts with the mostprevalent being 2.1 to 2.3 kb. Structural features include anintracellular domain sequence of about 52 amino acids, an extracellularregion of about 246 amino acids, and a hydrophobic presumptive membranespanning portion of about 20 amino acids. See Table 1 and SEQ. ID. NO:2. 499E9 exhibits structural motifs characteristic of a member of theTNF ligand family. Compare, e.g., with the CD40 ligand, OX40 ligand,TNF, NGF, and FAS. Table 1 illustrates the nucleic acid and predictedamino acid sequences for mouse 499E9.

TABLE 1 Mouse 499E9 nucleotide and predicted amino-acid sequence.Predicted intracellular domain sequence runs about from met1 to met49;residues 8 and 11 are potential tyrosine phosphorylation sites; atransmembrane sequence probably runs about from phe50 to leu69; and theextracellular domain probably runs about from tyr70 to asp316. See SEQID NO: 1 and 2. GCCAGGACCT CTGTGAACCG GTCGGGGCGG GGGCCGCCTG GCCGGGAGTCTGCTCGGCGG 60 TGGGTGGCCG AGGAAGGGAG AGAACGATCG CGGAGCAGGG CGCCCGAACTCCGGGCGCCG 120 CGCC ATG CGC CGG GCC AGC CGA GAC TAC GGC AAG TAC CTG CGCAGC TCG 169      Met Arg Arg Ala Ser Arg Asp Tyr Gly Lys Tyr Leu Arg SerSer        1               5                  10                  15 GAGGAG ATG GGC AGC GGC CCC GGC GTC CCA CAC GAG GGT CCG CTG CAC 217 Glu GluMet Gly Ser Gly Pro Gly Val Pro His Glu Gly Pro Leu His                 20                  25                  30 CCC GCG CCTTCT GCA CCG GCT CCG GCG CCG CCA CCC GCC GCC TCC CGC 265 Pro Ala Pro SerAla Pro Ala Pro Ala Pro Pro Pro Ala Ala Ser Arg             35                  40                  45 TCC ATG TTC CTGGCC CTC CTG GGG CTG GGA CTG GGC CAG GTG GTC TGC 313 Ser Met Phe Leu AlaLeu Leu Gly Leu Gly Leu Gly Gln Val Val Cys         50                  55                  60 AGC ATC GCT CTG TTCCTG TAC TTT CGA GCG CAG ATG GAT CCT AAC AGA 361 Ser Ile Ala Leu Phe LeuTyr Phe Arg Ala Gln Met Asp Pro Asn Arg     65                  70                  75 ATA TCA GAA GAC AGC ACTCAC TGC TTT TAT AGA ATC CTG AGA CTC CAT 409 Ile Ser Glu Asp Ser Thr HisCys Phe Tyr Arg Ile Leu Arg Leu His 80                  85                  90                  95 GAA AACGCA GGT TTG CAG GAC TCG ACT CTG GAG AGT GAA GAC ACA CTA 457 Glu Asn AlaGly Leu Gln Asp Ser Thr Leu Glu Ser Glu Asp Thr Leu                100                 105                 110 CCT GAC TCCTGC AGG AGG ATG AAA CAA GCC TTT CAG GGG GCC GTG CAG 505 Pro Asp Ser CysArg Arg Met Lys Gln Ala Phe Gln Gly Ala Val Gln            115                 120                 125 AAG GAA CTG CAACAC ATT GTG GGG CCA CAG CGC TTC TCA GGA GCT CCA 553 Lys Glu Leu Gln HisIle Val Gly Pro Gln Arg Phe Ser Gly Ala Pro        130                 135                 140 GCT ATG ATG GAA GGCTCA TGG TTG GAT GTG GCC CAG CGA GGC AAG CCT 601 Ala Met Met Glu Gly SerTrp Leu Asp Val Ala Gln Arg Gly Lys Pro    145                 150                 155 GAG GCC CAG CCA TTT GCACAC CTC ACC ATC AAT GCT GCC AGC ATC CCA 649 Gln Ala Gln Pro Phe Ala HisLeu Thr Ile Asn Ala Ala Ser Ile Pro160                 165                 170                 175 TCG GGTTCC CAT AAA GTC ACT CTG TCC TCT TGG TAC CAC GAT CGA GGC 697 Ser Gly SerHis Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly                180                 185                 190 TGG GCC AAGATC TCT AAC ATG ACG TTA AGC AAC GGA AAA CTA AGG GTT 745 Trp Ala Lys IleSer Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val            195                 200                 205 AAC CAA GAT GGCTTC TAT TAC CTG TAC GCC AAC ATT TGC TTT CGG CAT 793 Asn Gln Asp Gly PheTyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His        210                 215                 220 CAT GAA ACA TCG GGAAGC GTA CCT ACA GAC TAT CTT CAG CTG ATG GTG 841 His Glu Thr Ser Gly SerVal Pro Thr Asp Tyr Leu Gln Leu Met Val    225                 230                 235 TAT GTC GTT AAA ACC AGCATC AAA ATC CCA AGT TCT CAT AAC CTG ATG 889 Tyr Val Val Lys Thr Ser IleLys Ile Pro Ser Ser His Asn Leu Met240                 245                 250                 255 AAA GGAGGG AGC ACG AAA AAC TGG TCG GGC AAT TCT GAA TTC CAC TTT 937 Lys Gly GlySer Thr Lys Asn Trp Ser Gly Asn Ser Glu Phe His Phe                260                 265                 270 TAT TCC ATAAAT GTT GGG GGA TTT TTC AAG CTC CGA GCT GGT GAA GAA 985 Tyr Ser Ile AsnVal Gly Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu            275                 280                 285 ATT AGC ATT CAGGTG TCC AAC CCT TCC CTG CTG GAT CCG GAT CAA GAT 1033 Ile Ser Ile Gln ValSer Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp        290                 295                 300 GCG ACG TAC TTT GGGGCT TTC AAA GTT CAG GAC ATA GAC TGAGACTCAT 1082 Ala Thr Tyr Phe Gly AlaPhe Lys Val Gln Asp Ile Asp    305                 310                 315 TTCGTGGAAC ATTAGCATGGATGTCCTAGA TGTTTGGAAA CTTCTTAAAA AATGGATGAT 1142 GTCTATACAT GTGTAAGACTAGTAAGAGAC ATGGCCCACG GTGTATGAAA CTCACAGCCC 1202 TCTCTCTTGA GCCTGTACAGGTTGTGTATA TGTAAAGTCC ATAGGTGATG TTAGATTCAT 1262 GGTGATTACA CAACGGTTTTACAATTTTGT AATGATTTCC TAAGAATTGA ACCAGATTGG 1322 GAGAGGTATT CCGATGCTTATGAAAAACTT ACACGTGAGC TATGGAAGGG GGTCACAGTC 1382 TCTGGGTCTA ACCCCTGGACATGTGCCACT GAGAACCTTG AAATTAAGAA GATGCCATGT 1442 CATTGCAAAG AAATGATAGTGTGAAGGGTT AAGTTCTTTT GAATTGTTAC ATTGCGCTGG 1502 GACCTGCAAA TAAGTTCTTTTTTTCTAATG AGGAGAGAAA AATATATGTA TTTTTATATA 1562 ATGTCTAAAG TTATATTTCAGGTGTAATGT TTTCTGTGCA AAGTTTTGTA AATTATATTT 1622 GTGCTATAGT ATTTGATTCAAAATATTTAA AAATGTCTCA CTGTTGACAT ATTTAATGTT 1682 TTAAATGTAC AGATGTATTTAACTGGTGCA CTTTGTAATT CCCCTGAAGG TACTCGTAGC 1742 TAAGGGGGCA GAATACTGTTTCTGGTGACC ACATGTAGTT TATTTCTTTA TTCTTTTTAA 1802 CTTAATAGAG TCTTCAGACTTGTCAAAACT ATGCAAGCAA AATAAATAAA TAAAAATAAA 1862 ATGAATATCT TGAATAATAAGTAGGATGTT GGTCACCAGG TGCCTTTCAA ATTTAGAAGC 1922 TAATTGACTT TAGGAGCTGACATAGCCAAA AAGGATACAT AATAGGCTAC TGAAAATCTG 1982 TCAGGAGTAT TTATGCAATTATTGAACAGG TGTCTTTTTT TACAAGAGCT ACAAATTGTA 2042 AATTTTGTTT CTTTTTTTTCCCATAGAAAA TGTACTATAG TTTATCAGCC AAAAAACAAT 2102 CCACTTTTTA ATTTAGTGAAAGTTATTTTA TTATACTGTA CAATAAAAGC ATTGTTTCTG 2162 AATGGCATTT TTTGGTACTTAAAAATGGC 2191

TNF ligand family members have a conserved leucine residue correspondingto 205; a conserved glycine residue corresponding to residue 211; aconserved tyrosine residue corresponding to 216; a conserved glycineresidue corresponding to residue 277; a conserved leucine residuecorresponding to residue 282; a conserved phenylalanine residuecorresponding to 307; and a conserved glycine residue corresponding toresidue 308. The TNF ligand domain seems to run about from 205 (leu) to316 (asp). Glycosylation sites may be at 197 and 262. This cloneexhibits closes homology to a mouse TRAIL, which is implicated ininduction of apoptosis. Related family members include ligands for CD40and FAS, and lymphotoxin beta, tumor necrosis factor, etc.

By cDNA Southern analysis, it is clear that 499E9 is expressed in many Tcells, including Th1, Th2, 3 week polarized Th1 or Th2 cells, pre Tcells, and in Rag knock-out thymus cDNA libraries. Some weak signal fromdendritic cells may have been detected. Cells expressing 499E9 typicallycontain a main transcript of about 2.1 to 2.3 kb, but also containingother transcripts. Tissue distribution analysis suggests a positivesignal in brain, heart, kidney, liver, lung, spleen, and testis.Transcripts for 499E9 have not been detected in fibroblasts (L cells),monocytes (RAW264), naive T cells (CD4+, MEL14+, Br cells), macrophagecells, Nippo infected lung/liver/spleen, or Rag knock out organs (brain,heart, kidney, liver, lung, spleen, or testis).

The structural homology of 499E9 to the TNF ligand family suggestsfunction of this molecule. 499E9, as a T cell surface molecule, likelymodulates Ag-specific proliferative responses on effector cells, orinduction of apoptosis of those cells. 499E9 agonists, or antagonists,may also act as a co-stimulatory molecule for regulation of T cellmediated cell activation, and may in fact, cause a shift of T helpercell types, e.g., between Th1 and Th2. Thus, 499E9 or antagonists shouldbe useful in the treatment of abnormal immune disorders, e.g., T cellimmune deficiencies, chronic inflammation, or tissue rejection.

TNF ligand molecules typically modulate cell proliferation, viability,and differentiation. For example, TNF and FAS can kill cells expressingtheir respective receptors, including fibroblasts, liver cells, andlymphocytes. Some members of this class of ligands exhibit effects oncellular proliferation of cells expressing their respective receptors,e.g., B cells expressing CD40. These effects on proliferation may alsoeffect subsequent differentiation steps, and may lead, directly orindirectly, to changes in cytokine expression profiles.

The members of the TNF ligand family also exhibit costimulation effects,which may also regulate cellular differentiation or apoptosis. Receptorexpressing cells may be protected from activation induced cell death(AICD) or apoptosis. For example, CD40 ligand can have effects on T andB lymphocytes.

The embodiment characterized herein is from mouse, but other primate,e.g., human, variants will exist. Additional sequences for proteins inother mammalian species, e.g., primates and rodents, will also beavailable. See below. The descriptions below are directed, for exemplarypurposes, to a mouse 499E9, but are likewise applicable to relatedembodiments from other species.

The mouse 499E9 protein is a protein which exhibits structural featurescharacteristic of a cell surface antigen, e.g., a TNF ligand familymember. The protein is easily detected on particular cell types, othersexpress lesser amounts. The 499E9 antigen should be present in theidentified tissue types and the interaction of the antigen with itsbinding partner should be important for mediating various aspects ofcellular physiology or development, as described.

II. Purified 499E9

Mouse 499E9 amino acid sequence is shown in SEQ ID NO: 2. These aminoacid sequences, provided amino to carboxy, are important in providingsequence information in the antigen allowing for distinguishing theprotein from other proteins and exemplifying numerous variants.Moreover, the peptide sequences allow preparation of peptides togenerate antibodies to recognize such segments, and nucleotide sequencesallow preparation of oligonucleotide probes, both of which arestrategies for detection or isolation, e.g., cloning, of genes or cDNAsencoding such sequences.

As used herein, the term “mouse 499E9” shall encompass, when used in aprotein context, a protein having amino acid sequence shown in SEQ IDNO: 2, or a significant fragment of such a protein, or another highlyhomologous protein derived from mouse. These binding components, e.g.,antibodies, typically bind to a 499E9 with high affinity, e.g., at leastabout 100 nM, usually better than about 30 nm, preferably better thanabout 10 μM, and more preferably at better than about 3 nM. Homologousproteins would be found in mammalian species other than mouse, e.g.,primates or rodents. Non-mammalian species should also possessstructurally or functionally related genes and proteins, e.g., birds oramphibians.

The term “polypeptide” as used herein includes a significant fragment orsegment, and encompasses a stretch of amino acid residues of at leastabout 8 amino acids, generally at least about 12 amino acids, typicallyat least about 16 amino acids, preferably at least about 20 amino acids,and, in particularly preferred embodiments, at least about 30 or moreamino acids.

The term “binding composition” refers to molecules that bind withspecificity to 499E9, e.g., in a cell adhesion pairing type fashion, oran antibody-antigen interaction. It also includes compounds, e.g.,proteins, which specifically associate with 499E9, including in anatural physiologically relevant protein-protein interaction, eithercovalent or non-covalent. The molecule may be a polymer, or chemicalreagent. A functional analog may be an antigen with structuralmodifications, or it may be a molecule which has a molecular shape whichinteracts with the appropriate binding determinants. The compounds mayserve as agonists or antagonists of the binding interaction, see, e.g.,Goodman, et al. (eds.) (1990) Goodman & Gilman's: The PharmacologicalBases of Therapeutics (8th ed.), Pergamon Press.

Substantially pure typically means that the protein is free from othercontaminating proteins, nucleic acids, or other biologicals derived fromthe original source organism. Purity may be assayed by standard methods,typically by weight, and will ordinarily be at least about 40% pure,generally at least about 50% pure, often at least about 60% pure,typically at least about 80% pure, preferably at least about 90% pure,and in most preferred embodiments, at least about 95% pure. Carriers orexcipients will often be added.

Solubility of a polypeptide or fragment depends upon the environment andthe polypeptide. Many parameters affect polypeptide solubility,including temperature, electrolyte environment, size and molecularcharacteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. For diagnostic purposes, the temperature will usuallybe about room temperature or warmer, but less than the denaturationtemperature of components in the assay. For therapeutic purposes, thetemperature will usually be body temperature, typically about 37° C. forhumans and mice, though under certain situations the temperature may beraised or lowered in situ or in vitro.

The size and structure of the polypeptide should generally be in asubstantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents in a manner which approximates natural lipid bilayerinteractions.

The solvent and electrolytes will usually be a biologically compatiblebuffer, of a type used for preservation of biological activities, andwill usually approximate a physiological aqueous solvent. Usually thesolvent will have a neutral pH, typically between about 5 and 10, andpreferably about 7.5. On some occasions, one or more detergents will beadded, typically a mild non-denaturing one, e.g., CHS (cholesterylhemisuccinate) or CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or a low enough concentration as to avoid significantdisruption of structural or physiological properties of the protein

III. Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence identity with the amino acid sequence of the 499E9.The variants include species, polymorphic, or allelic variants.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) Chapter One in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group, Madison, Wis. Sequence identity changes whenconsidering conservative substitutions as matches. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. Homologous amino acid sequencesare typically intended to include natural polymorphic or allelic andinterspecies variations in each respective protein sequence. Typicalhomologous proteins or peptides will have from 25-100% identity (if gapscan be introduced), to 50-100% identity (if conservative substitutionsare included) with the amino acid sequence of the 499E9. Identitymeasures will be at least about 35%, generally at least about 40%, oftenat least about 50%, typically at least about 60%, usually at least about70%, preferably at least about 80%, and more preferably at least about90%.

The isolated 499E9 DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of nucleotide stretches. These modifications result in novelDNA sequences which encode these antigens, their derivatives, orproteins having similar physiological, immunogenic, antigenic, or otherfunctional activity. These modified sequences can be used to producemutant antigens or to enhance expression. Enhanced expression mayinvolve gene amplification, increased transcription, increasedtranslation, and other mechanisms. “Mutant 499E9” encompasses apolypeptide otherwise falling within the sequence identity definition ofthe 499E9 as set forth above, but having an amino acid sequence whichdiffers from that of 499E9 as normally found in nature, whether by wayof deletion, substitution, or insertion. This generally includesproteins having significant identity with a protein having sequence ofSEQ ID NO: 2, and as sharing various biological activities, e.g.,antigenic or immunogenic, with those sequences, and in preferredembodiments contain most of the full length disclosed sequences. Fulllength sequences will typically be preferred, though truncated versions,e.g., soluble constructs and intact domains, will also be useful,likewise, genes or proteins found from natural sources are typicallymost desired. Similar concepts apply to different 499E9 proteins,particularly those found in various warm blooded animals, e.g., mammalsand birds. These descriptions are generally meant to encompass all 499E9proteins, not limited to the particular mouse embodiments specificallydiscussed.

499E9 mutagenesis can also be conducted by making amino acid insertionsor deletions. Substitutions, deletions, insertions, or any combinationsmay be generated to arrive at a final construct. Insertions includeamino- or carboxy-terminal fusions Random mutagenesis can be conductedat a target codon and the expressed mutants can then be screened for thedesired activity. Methods for making substitution mutations atpredetermined sites in DNA having a known sequence are well known in theart, e.g., by M13 primer mutagenesis or polymerase chain reaction (PCR)techniques. See, e.g., Sambrook, et al. (1989); Ausubel, et al. (1987and Supplements); and Kunkel, et al. (1987) Methods in Enzymol.154:367-382.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. A similar conceptapplies to heterologous nucleic acid sequences. Fusion proteins will beuseful as sources for cleaving, separating, and purifying portionsthereof.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, target-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263.15985-15992.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques.

IV. Functional Variants

The blocking of physiological response to 499E9s may result from theinhibition of binding of the antigen to its binding partner, e.g.,another of itself, likely through competitive inhibition. Thus, in vitroassays of the present invention will often use isolated protein,membranes from cells expressing a membrane associated recombinant 499E9,soluble fragments comprising antigen binding segments of these proteins,or fragments attached to solid phase substrates. These assays will alsoallow for the diagnostic determination of the effects of either bindingsegment mutations and modifications, or antigen mutations andmodifications, e.g., 499E9 analogs.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to antigen or bindingfragments compete with a test compound for binding to the protein, e.g.,of natural protein sequence.

“Derivatives” of 499E9 antigens include amino acid sequence mutants fromnaturally occurring forms, glycosylation variants, and covalent oraggregate conjugates with other chemical moieties. Covalent derivativescan be prepared by linkage of functionalities to groups which are foundin 499E9 amino acid side chains or at the N- or C-termini, e.g., bystandard means. See, e.g., Lundblad and Noyes (1988) Chemical Reagentsfor Protein Modification, vols. 1-2, CRC Press, Inc., Boca Raton, Fla.;Hugli (ed.) (1989) Techniques in Protein Chemistry, Academic Press, SanDiego, Calif.; and Wong (1991) Chemistry of Protein Conjugation andCross Linking, CRC Press, Boca Raton, Fla.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. See, e.g.,Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versionsof the peptides with the same primary amino acid sequence which haveother minor modifications, including phosphorylated amino acid residues,e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Fusion polypeptides between 499E9s and other homologous or heterologousproteins are also provided. Many cytokine receptors or other surfaceproteins are multimeric, e.g., homodimeric entities, and a repeatconstruct may have various advantages, including lessened susceptibilityto proteolytic cleavage. Typical examples are fusions of a reporterpolypeptide, e.g., luciferase, with a segment or domain of a protein,e.g., a receptor-binding segment, so that the presence or location ofthe fused ligand may be easily determined. See, e.g., Dull, et al., U.S.Pat. No. 4,859,609. Other gene fusion partners include bacterialβ-galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcoholdehydrogenase, yeast alpha mating factor, and detection or purificationtags such as a FLAG sequence of His6 sequence. See, e.g., Godowski, etal. (1988) Science 241:812-816.

Fusion peptides will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, e.g., inSambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.),vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al. (eds.)(1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.Techniques for synthesis of polypeptides are described, e.g., inMerrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986)Science 232: 341347; Atherton, et al. (1989) Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford; and Grant (1992)Synthetic Peptides: A User's Guide, W.H. Freeman, N.Y.

This invention also contemplates the use of derivatives of 499E9s otherthan variations in amino acid sequence or glycosylation. Suchderivatives may involve covalent or aggregative association withchemical moieties. Covalent or aggregative derivatives will be useful asimmunogens, as reagents in immunoassays, or in purification methods suchas for affinity purification of binding partners, e.g., other antigens.A 499E9 can be immobilized by covalent bonding to a solid support suchas cyanogen bromide-activated SEPHAROSE, by methods which are well knownin the art, or adsorbed onto polyolefin surfaces, with or withoutglutaraldehyde cross-linking, for use in the assay or purification ofanti-499E9 antibodies or an alternative binding composition. The 499E9scan also be labeled with a detectable group, e.g., for use in diagnosticassays. Purification of 499E9 may be effected by an immobilized antibodyor complementary binding partner.

A solubilized 499E9 or fragment of this invention can be used as animmunogen for the production of antisera or antibodies specific forbinding to the antigen or fragments thereof. Purified antigen can beused to screen monoclonal antibodies or antigen-binding fragments,encompassing antigen binding fragments of natural antibodies, e.g., Fab,Fab′, F(ab)₂, etc. Purified 499E9s can also be used as a reagent todetect antibodies generated in response to the presence of elevatedlevels of the antigen or cell fragments containing the antigen, both ofwhich may be diagnostic of an abnormal or specific physiological ordisease condition. This invention contemplates antibodies raised againstamino acid sequences encoded by nucleotide sequence shown in SEQ ID NO:1, or fragments of proteins containing it. In particular, this inventioncontemplates antibodies having binding affinity to or being raisedagainst specific fragments which are predicted to lie outside of thelipid bilayer, both extracellular or intracellular.

The present invention contemplates the isolation of additional closelyrelated species variants. Southern and Northern blot analysis shouldestablish that similar genetic entities exist in other mammals. It islikely that 499E9s are widespread in species variants, e.g., rodents,lagomorphs, carnivores, artiodactyla, perissodactyla, and primates.

The invention also provides means to isolate a group of related antigensdisplaying both distinctness and similarities in structure, expression,and function. Elucidation of many of the physiological effects of themolecules will be greatly accelerated by the isolation andcharacterization of additional distinct species variants of them. Inparticular, the present invention provides useful probes for identifyingadditional homologous genetic entities in different species.

The isolated genes will allow transformation of cells lacking expressionof a corresponding 499E9, e.g., either species types or cells which lackcorresponding antigens and exhibit negative background activity. Thisshould allow analysis of the function of 499E9 in comparison tountransformed control cells.

Dissection of critical structural elements which effect the variousactivation or differentiation functions mediated through these antigensis possible using standard techniques of modern molecular biology,particularly in comparing members of the related class. See, e.g., thehomolog-scanning mutagenesis technique described in Cunningham, et al.(1989) Science 243:1339-1336; and approaches used in O'Dowd, et al.(1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, et al. (1990)EMBO J. 9:4381-4390.

Intracellular functions would probably involve segments of the antigenwhich are normally accessible to the cytosol. However, proteininternalization may occur under certain circumstances, and interactionbetween intracellular components and “extracellular” segments may occur.The specific segments of interaction of 499E9 with other intracellularcomponents may be identified by mutagenesis or direct biochemical means,e.g., cross-linking or affinity methods. Structural analysis bycrystallographic or other physical methods will also be applicable.Further investigation of the mechanism of signal transduction willinclude study of associated components which may be isolatable byaffinity methods or by genetic means, e.g., complementation analysis ofmutants.

Further study of the expression and control of 499E9 will be pursued.The controlling elements associated with the antigens should exhibitdifferential physiological, developmental, tissue specific, or otherexpression patterns. Upstream or downstream genetic regions, e.g.,control elements, are of interest. In particular, physiological ordevelopmental variants, e.g., multiple alternatively processed forms ofthe antigen have been found. See, e.g., SEQ ID NO: 1. Thus, differentialsplicing of message may lead to an assortment of membrane bound forms,soluble forms, and modified versions of antigen.

Structural studies of the antigens will lead to design of new antigens,particularly analogs exhibiting agonist or antagonist properties on themolecule. This can be combined with previously described screeningmethods to isolate antigens exhibiting desired spectra of activities.

V. Antibodies

Antibodies can be raised to various 499E9s, including species,polymorphic, or allelic variants, and fragments thereof, both in theirnaturally occurring forms and in their recombinant forms. Additionally,antibodies can be raised to 499E9s in either their active forms or intheir inactive forms, including native or denatured versions.Anti-idiotypic antibodies are also contemplated.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the antigens can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective 499E9s, or screened for agonistic orantagonistic activity, e.g., mediated through the antigen or its bindingpartner. Antibodies may be agnostic or antagonistic, e.g., by stericallyblocking ligand binding. These monoclonal antibodies will usually bindwith at least a K_(D) of about 1 mM, more usually at least about 300 μM,typically at least about 100 μM, more typically at least about 30 μM,preferably at least about 10 μM, and more preferably at least about 3 μMor better.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for ability to bind to the antigens without inhibiting bindingby a partner. As neutralizing antibodies, they can be useful incompetitive binding assays. They will also be useful in detecting orquantifying 499E9 protein or its binding partners. See, e.g., Chan (ed.)(1987) Immunology: A Practical Guide, Academic Press, Orlando, Fla.;Price and Newman (eds.) (1991) Principles and Practice of Immunoassay,Stockton Press, N.Y.; and Ngo (ed.) (1988) Nonisotopic Immunoassay,Plenum Press, N.Y. Cross absorptions or other tests will identifyantibodies which exhibit various spectra of specificities, e.g., uniqueor shared species specificities.

Further, the antibodies, including antigen binding fragments, of thisinvention can be potent antagonists that bind to the antigen and inhibitfunctional binding or inhibit the ability of a binding partner to elicita biological response. They also can be useful as non-neutralizingantibodies and can be coupled to toxins or radionuclides so that whenthe antibody binds to antigen, a cell expressing it, e.g., on itssurface, is killed. Further, these antibodies can be conjugated to drugsor other therapeutic agents, either directly or indirectly by means of alinker, and may effect drug targeting.

Antigen fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. An antigen and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner (1962) Specificityof Serological Reactions, Dover Publications, New York; Williams, et al.(1967) Methods in Immunology and Immunochemistry, vol. 1, AcademicPress, New York; and Harlow and Lane (1988) Antibodies: A LaboratoryManual, CSH Press, NY, for descriptions of methods of preparingpolyclonal antisera.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.), Academic Press, New York; and particularly in Kohler andMilstein (1975) in Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore, et al.,U.S. Pat. No. 4,642,334; and Queen, et al. (1989) Proc. Nat'l Acad. Sci.USA 86:10029-10033.

The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support. See, e.g., Wilchek et al.(1984) Meth. Enzymol. 104:3-55.

Antibodies raised against each 499E9 will also be useful to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

VI. Nucleic Acids

The described peptide sequences and the related reagents are useful indetecting, isolating, or identifying a DNA clone encoding 499E9, e.g.,from a natural source. Typically, it will be useful in isolating a genefrom mammal, and similar procedures will be applied to isolate genesfrom other species, e.g., warm blooded animals, such as birds andmammals. Cross hybridization will allow isolation of 499E9 from otherspecies. A number of different approaches should be available tosuccessfully isolate a suitable nucleic acid clone.

The purified protein or defined peptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein can be presented to an immune system to generatemonoclonal or polyclonal antibodies. See, e.g., Coligan (1991) CurrentProtocols in Immunology Wiley/Greene; and Harlow and Lane (1989)Antibodies: A Laboratory Manual, Cold Spring Harbor Press.Alternatively, the 499E9 can be used as a specific binding reagent, andadvantage can be taken of its specificity of binding, much like anantibody would be used.

For example, the specific binding composition could be used forscreening of an expression library made from a cell line which expressesa 499E9. The screening can be standard staining of surface expressedantigen, or by panning. Screening of intracellular expression can alsobe performed by various staining or immunofluorescence procedures. Thebinding compositions could be used to affinity purify or sort out cellsexpressing the protein.

The peptide segments can also be used to predict appropriateoligonucleotides to screen a library. The genetic code can be used toselect appropriate oligonucleotides useful as probes for screening. See,e.g., SEQ ID NO: 1. In combination with polymerase chain reaction (PCR)techniques, synthetic oligonucleotides will be useful in selectingcorrect clones from a library. Complementary sequences will also be usedas probes, primers, or antisense strands. Based upon identification ofthe likely extracellular domain, various fragments should beparticularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

This invention contemplates use of isolated DNA or fragments to encode abiologically active corresponding 499E9 polypeptide. In addition, thisinvention covers isolated or recombinant DNA which encodes abiologically active protein or polypeptide which is capable ofhybridizing under appropriate conditions with the DNA sequencesdescribed herein. Said biologically active protein or polypeptide can bean intact antigen, or fragment, and have an amino acid sequencedisclosed in, e.g., SEQ ID NO: 1. Further, this invention covers the useof isolated or recombinant DNA, or fragments thereof, which encodeproteins which are homologous to a 499E9 or which was isolated usingcDNA encoding a 499E9 as a probe. The isolated DNA can have therespective regulatory sequences in the 5′ and 3′ flanks, e.g.,promoters, enhancers, poly-A addition signals, and others.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other componentswhich naturally accompany a native sequence, e.g., ribosomes,polymerases, and/or flanking genomic sequences from the originatingspecies. The term embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.Generally, the nucleic acid will be in a vector or fragment less thanabout 50 kb, usually less than about 30 kb, typically less than about 10kb, and preferably less than about 6 kb.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain minor heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, e.g., products made by transforming cells with anyunnaturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site.

Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a single genetic entity comprising adesired combination of functions not found in the commonly availablenatural forms. Restriction enzyme recognition sites are often the targetof such artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. Specifically included are synthetic nucleic acids which, bygenetic code redundancy, encode polypeptides similar to fragments ofthese antigens, and fusions of sequences from various different speciesvariants.

A significant “fragment” in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least about 22nucleotides, ordinarily at least about 29 nucleotides, more often atleast about 35 nucleotides, typically at least about 41 nucleotides,usually at least about 47 nucleotides, preferably at least about 55nucleotides, and in particularly preferred embodiments will be at leastabout 60 or more nucleotides.

A DNA which codes for a 499E9 protein will be particularly useful toidentify genes, mRNA, and cDNA species which code for related orhomologous proteins, as well as DNAs which code for homologous proteinsfrom different species. There are likely homologs in other species,including primates, rodents, and birds. Various 499E9 proteins should behomologous and are encompassed herein. However, even genes encodingproteins that have a more distant evolutionary relationship to theantigen can readily be isolated under appropriate conditions using thesesequences if they are sufficiently homologous. Primate 499E9 proteinsare of particular interest, Recombinant clones derived from the genomicsequences, e.g., containing introns, will be useful for transgenicstudies, including, e.g., transgenic cells and organisms, and for genetherapy. See, e.g., Goodnow (1992) “Transgenic Animals” in Roitt (ed.)Encyclopedia of Immunology, Academic Press, San Diego, pp. 1502-1504;Travis (1992) Science 256:1392-1394; Kuhn, et al. (1991) Science254:707-710; Capecchi (1989) Science 244:1288; Robertson (1987) (ed.)Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, IRLPress, Oxford; and Rosenberg (1992) J. Clinical Oncology 10:180-199.

Substantial homology in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 50% of thenucleotides, generally at least about 58%, ordinarily at least about65%, often at least about 71%, typically at least about 77%, usually atleast about 85%, preferably at least about 95 to 98% or more, and inparticular embodiments, as high as about 99% or more of the nucleotides.Alternatively, substantial homology exists when the segments willhybridize under selective hybridization conditions, to a strand, or itscomplement, typically using a sequence of 499E9, e.g., in SEQ ID NO: 1.Typically, selective hybridization will occur when there is at leastabout 55% homology over a stretch of at least about 30 nucleotides,preferably at least about 75% over a stretch of about 25 nucleotides,and most preferably at least about 90% over about 20 nucleotides. See,Kanehisa (1984) Nuc. Acids Res. 12:203-213. The length of homologycomparison, as described, may be over longer stretches, and in certainembodiments will be over a stretch of at least about 17 nucleotides,usually at least about 28 nucleotides, typically at least about 40nucleotides, and preferably at least about 75 to 100 or morenucleotides.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters, typically those controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., usually in excess ofabout 37° C., typically in excess of about 55° C., preferably in excessof about 70° C. Stringent salt conditions will ordinarily be less thanabout 1000 mM, usually less than about 400 mM, typically less than about250 mM, preferably less than about 150 mM. However, the combination ofparameters is much more important than the measure of any singleparameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370.

499E9 from other mammalian species can be cloned and isolated bycross-species hybridization of closely related species. Homology may berelatively low between distantly related species, and thus hybridizationof relatively closely related species is advisable. Alternatively,preparation of an antibody preparation which exhibits less speciesspecificity may be useful in expression cloning approaches.

VII. Making 499E9; Mimetics

DNA which encodes the 499E9 or fragments thereof can be obtained bychemical synthesis, screening cDNA libraries, or screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.See, e.g., Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170; Gublerand Hoffman (1983) Gene 25:263-269; and Glover (ed.) (1984) DNA Cloning:A Practical Approach, IRL Press, Oxford. Alternatively, the sequencesprovided herein provide useful PCR primers or allow synthetic or otherpreparation of suitable genes encoding a 499E9; including, naturallyoccurring embodiments.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length 499E9 or fragments which can in turn, e.g.,be used to generate polyclonal or monoclonal antibodies; for bindingstudies; for construction and expression of modified molecules; and forstructure/function studies.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. See, e.g.,Pouwels, et al. (1985 and Supplements) Cloning Vectors: A LaboratoryManual, Elsevier, N.Y.; and Rodriguez, et al. (1988) (eds.) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Buttersworth,Boston, Mass.

For purposes of this invention, DNA sequences are operably linked whenthey are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression. See e.g., Rodriguez, et al., Chapter 10, pp. 205-236; Balbasand Bolivar (1990) Methods in Enzymology 185:14-37; and Ausubel, et al.(1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.

Representative examples of suitable expression vectors include pcDNA1;pCD, see Okayama, et al. (1985) Mol. Cell. Biol. 5:1136-1142; pMC1neoPoly-A, see Thomas, et al. (1987) Cell 51:503-512; and a baculovirusvector such as pAC 373 or pAC 610.

See, e.g., Miller (1988) Ann. Rev. Microbiol. 42:177-199.

It will often be desired to express a 499E9 polypeptide in a systemwhich provides a specific or defined glycosylation pattern. See, e.g.,Luckow and Summers (1988) Bio/Technology 6:47-55; and Kaufman (1990)Meth. Enzymol. 185:487-511.

The 499E9, or a fragment thereof, may be engineered to be phosphatidylinositol (PI) linked to a cell membrane, but can be removed frommembranes by treatment with a phosphatidyl inositol cleaving enzyme,e.g., phosphatidyl inositol phospholipase-C. This releases the antigenin a biologically active form, and allows purification by standardprocedures of protein chemistry. See, e.g., Low (1989) Biochim. Biophys.Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner,et al. (1991) J. Cell Biol. 114:1275-1283.

Now that the 499E9 has been characterized, fragments or derivativesthereof can be prepared by conventional processes for synthesizingpeptides. These include processes such as are described in Stewart andYoung (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co.,Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice of PeptideSynthesis, Springer-verlag, New York; Bodanszky (1984) The Principles ofPeptide Synthesis, Springer-Verlag, New York; and Villafranca (ed.)(1991) Techniques in Protein Chemistry II, Academic Press, San Diego,Calif.

VIII. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in thegeneral description for T cell mediated conditions, or below in thedescription of kits for diagnosis.

This invention also provides reagents with significant therapeuticvalue. The 499E9 (naturally occurring or recombinant), fragmentsthereof, and antibodies thereto, along with compounds identified ashaving binding affinity to 499E9, should be useful in the treatment ofconditions associated with abnormal physiology or development, includingabnormal proliferation, e.g., cancerous conditions, or degenerativeconditions. In particular, modulation of development of lymphoid cellswill be achieved by appropriate therapeutic treatment using thecompositions provided herein. For example, a disease or disorderassociated with abnormal expression or abnormal signaling by a 499E9should be a likely target for an agonist or antagonist of the antigen.The antigen plays a role in regulation or development of hematopoieticcells, e.g., lymphoid cells, which affect immunological responses, e.g.,autoimmune disorders.

In particular, the antigen will likely provide a costimulatory signal tocell activation. Thus, the 499E9 will likely modulate T cell mediatedinteractions with other cell types, e.g., cells which possess a receptortherefor. These interactions would lead, in particular contexts, tomodulation of cell growth, cytokine synthesis by those or other cells,or development of particular effector cells.

Moreover, the 499E9 or antagonists could redirect T cell responses,e.g., between Th1 and Th2 polarization, or with Th0 cells. Among theseagonists should be various antibodies which recognize the appropriateepitopes, e.g., which mimic binding of 499E9 to its receptor.Alternatively, they may bind to epitopes which sterically can blockreceptor binding.

Antagonists of 499E9, such as the naturally occurring secreted form of499E9 or blocking antibodies, may also be useful, They may provide aselective and powerful way to modulate immune responses in abnormalsituations, e.g., autoimmune disorders, including rheumatoid arthritis,systemic lupus erythematosis (SLE), Hashimoto's autoimmune thyroiditis,as well as acute and chronic inflammatory responses in which T cellactivation, expansion, and/or immunological T cell memory play animportant role. See also Samter, et al. (eds) Immunological Diseasesvols. 1 and 2, Little, Brown and Co. Regulation of T cell activation,expansion, and/or cytokine release by the naturally occurring secretedform of 499E9, or an antagonist thereof, may be effected.

In addition, certain combination compositions with other modulators of Tcell signaling would be useful. Such other signaling molecules includeTcR reagents, CD40, CD40L, CTLA-S, CD28, SLAM, FAS, and their respectiveantagonists.

Various abnormal conditions are known in each of the cell types shown topossess 499E9 mRNA by Northern blot analysis. See Berkow (ed.) The MerckManual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; Thorn, etal. Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.; andWeatherall, et al. (eds.) Oxford Textbook of Medicine, Oxford UniversityPress, Oxford. Many other medical conditions and diseases involve Tcells or are T cell mediated, and many of these will be responsive totreatment by an agonist or antagonist provided herein. See, e.g., Stitesand Terr (eds; 1991) Basic and Clinical Immunology Appleton and Lange,Norwalk, Conn.; and Samter, et al. (eds) Immunological Diseases Little,Brown and Co. These problems should be susceptible to prevention ortreatment using compositions provided herein.

499E9 antibodies can be purified and then administered to a patient,veterinary or human. These reagents can be combined for therapeutic usewith additional active or inert ingredients, e.g., in conventionalpharmaceutically acceptable carriers or diluents, e.g., immunogenicadjuvants, along with physiologically innocuous stabilizers, excipients,or preservatives. These combinations can be sterile filtered and placedinto dosage forms as by lyophilization in dosage vials or storage instabilized aqueous preparations. This invention also contemplates use ofantibodies or binding fragments thereof, including forms which are notcomplement binding.

Drug screening using 499E9 or fragments thereof can be performed toidentify compounds having binding affinity to or other relevantbiological effects on 499E9 functions, including isolation of associatedcomponents. Subsequent biological assays can then be utilized todetermine if the compound has intrinsic stimulating activity or is ablocker or antagonist in that it blocks the activity of the antigen,e.g., mutein antagonists. Likewise, a compound having intrinsicstimulating activity can activate the signal pathway and is thus anagonist in that it simulates the activity of 499E9. This inventionfurther contemplates the therapeutic use of blocking antibodies to 499E9as antagonists and of stimulatory molecules, e.g., muteins, as agonists.This approach should be particularly useful with other 499E9 speciesvariants.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17thed. (1990), Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 μM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous or long term administration. See, e.g., Langer (1990) Science249:1527-1533.

499E9, fragments thereof, and antibodies to it or its fragments,antagonists, and agonists, may be administered directly to the host tobe treated or, depending on the size of the compounds, it may bedesirable to conjugate them to carrier proteins such as ovalbumin orserum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, topical, or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. See, e.g., Gilman, et al.(eds.) (1990) Goodman and Gilman's: The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, etal. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications,Dekker, New York; Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Tablets, Dekker, New York; and Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York. Thetherapy of this invention may be combined with or used in associationwith other agents, e.g., other modulators of T cell activation, e.g.,CD40, CD40 ligand, CD28, CTLA-4, B7, B70, SLAM, T cell receptorsignaling entities, or their respective antagonists.

Both the naturally occurring and the recombinant form of the 499E9s ofthis invention are particularly useful in kits and assay methods whichare capable of screening compounds for binding activity to the proteins.Several methods of automating assays have been developed in recent yearsso as to permit screening of tens of thousands of compounds in a shortperiod. See, e.g., Fodor, et al. (1991) Science 251:767-773, whichdescribes means for testing of binding affinity by a plurality ofdefined polymers synthesized on a solid substrate. The development ofsuitable assays can be greatly facilitated by the availability of largeamounts of purified, soluble 499E9 as provided by this invention.

Other methods can be used to determine the critical residues in the499E9-499E9 receptor interactions. Mutational analysis can be performed,e.g., see Somoza, et al. (1993) J. Exptl. Med. 178:549-558, to determinespecific residues critical in the interaction and/or signaling. Bothextracellular domains, involved in the homophilic interaction, orintracellular domain, which provides interactions important inintracellular signaling.

For example, antagonists can normally be found once the antigen has beenstructurally defined, e.g., by tertiary structure data. Testing ofpotential interacting analogs is now possible upon the development ofhighly automated assay methods using a purified 499E9. In particular,new agonists and antagonists will be discovered by using screeningtechniques described herein. Of particular importance are compoundsfound to have a combined binding affinity for a spectrum of 499E9molecules, e.g., compounds which can serve as antagonists for speciesvariants of 499E9.

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing a 499E9. Cells may be isolated which express a 499E9 inisolation from other molecules. Such cells, either in viable or fixedform, can be used for standard binding partner binding assays. See also,Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990)Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto a 499E9 and is described in detail in Geysen, European PatentApplication 84/03564, published on Sep. 13, 1984. First, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, e.g., plastic pins or some other appropriate surface, seeFodor, et al. (1991). Then all the pins are reacted with solubilized,unpurified or solubilized, purified 499E9, and washed. The next stepinvolves detecting bound 499E9.

Rational drug design may also be based upon structural studies of themolecular shapes of the 499E9 and other effectors or analogs. Effectorsmay be other proteins which mediate other functions in response tobinding, or other proteins which normally interact with 499E9. One meansfor determining which sites interact with specific other proteins is aphysical structure determination, e.g., x-ray crystallography or 2dimensional NMR techniques. These will provide guidance as to whichamino acid residues form molecular contact regions. For a detaileddescription of protein structural determination, see, e.g., Blundell andJohnson (1976) Protein Crystallography, Academic Press, New York.

IX. Kits

This invention also contemplates use of 499E9 proteins, fragmentsthereof, peptides, and their fusion products in a variety of diagnostickits and methods for detecting the presence of another 499E9 or bindingpartner. Typically the kit will have a compartment containing either adefined 499E9 peptide or gene segment or a reagent which recognizes oneor the other, e.g., 499E9 fragments or antibodies.

A kit for determining the binding affinity of a test compound to a 499E9would typically comprise a test compound; a labeled compound, forexample a binding partner or antibody having known binding affinity for499E9; a source of 499E9 (naturally occurring or recombinant); and ameans for separating bound from free labeled compound, such as a solidphase for immobilizing the molecule. Once compounds are screened, thosehaving suitable binding affinity to the antigen can be evaluated insuitable biological assays, as are well known in the art, to determinewhether they act as agonists or antagonists to the 499E9 signalingpathway. The availability of recombinant 499E9 polypeptides also providewell defined standards for calibrating such assays.

A preferred kit for determining the concentration of, e.g., a 499E9 in asample would typically comprise a labeled compound, e.g., bindingpartner or antibody, having known binding affinity for the antigen, asource of antigen (naturally occurring or recombinant) and a means forseparating the bound from free labeled compound, e.g., a solid phase forimmobilizing the 499E9. Compartments containing reagents, andinstructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for the 499E9or fragments are useful in diagnostic applications to detect thepresence of elevated levels of 499E9 and/or its fragments. Suchdiagnostic assays can employ lysates, live cells, fixed cells,immunofluorescence, cell cultures, body fluids, and further can involvethe detection of antigens related to the antigen in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-binding partner complex) or heterogeneous (witha separation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525; Harlow andLane (1980) Antibodies: A Laboratory Manual, CSH Press, NY; and Coligan,et al. (eds.) (1993) Current Protocols in Immunology, Greene and Wiley,NY.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against a 499E9, as such may be diagnostic of variousabnormal states. For example, overproduction of 499E9 may result inproduction of various immunological reactions which may be diagnostic ofabnormal physiological states, particularly in proliferative cellconditions such as cancer or abnormal activation or differentiation.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or binding partner, or labeled499E9 is provided. This is usually in conjunction with other additives,such as buffers, stabilizers, materials necessary for signal productionsuch as substrates for enzymes, and the like. Preferably, the kit willalso contain instructions for proper use and disposal of the contentsafter use. Typically the kit has compartments for each useful reagent.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium providingappropriate concentrations of reagents for performing the assay.

Many of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification or may be modified ina variety of ways. For example, labeling may be achieved by covalentlyor non-covalently joining a moiety which directly or indirectly providesa detectable signal. In any of these assays, the binding partner, testcompound, 499E9, or antibodies thereto can be labeled either directly orindirectly. Possibilities for direct labeling include label groups:radiolabels such as 125j, enzymes (U.S. Pat. No. 3,645,090) such asperoxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat.No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups.

There are also numerous methods of separating the bound from the free499E9, or alternatively the bound from the free test compound. The 499E9can be immobilized on various matrixes followed by washing. Suitablematrixes include plastic such as an ELISA plate, filters, and beads.See, e.g., Coligan, et al. (eds.) (1993) Current Protocols inImmunology, Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle, et al. (1984)Clin. Chem. 30:1457-1461, and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,678.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence of a499E9. These sequences can be used as probes for detecting levels of the499E9 message in samples from patients suspected of having an abnormalcondition, e.g., cancer or developmental problem. Since the antigen is amarker for activation, it may be useful to determine the numbers ofactivated T cells to determine, e.g., when additional suppression may becalled for. The preparation of both RNA and DNA nucleotide sequences,the labeling of the sequences, and the preferred size of the sequenceshas received ample description and discussion in the literature. See,e.g., Langer-Safer, et al. (1982) Proc. Nat'l. Acad. Sci. 79:4381-4385;Caskey (1987) Science 236:962-967; and Wilchek et al. (1988) Anal.Biochem. 171:1-32.

Diagnostic kits which also test for the qualitative or quantitativepresence of other markers are also contemplated. Diagnosis or prognosismay depend on the combination of multiple indications used as markers.Thus, kits may test for combinations of markers. See, e.g., Viallet, etal. (1989) Progress in Growth Factor Res. 1:89-97. Other kits may beused to evaluate T cell subsets.

X. Methods for Isolating 499E9 Specific Binding Partners

The 499E9 protein should interact with a receptor based, e.g., upon itssimilarity in structure and function to other cell surface antigensexhibiting similar structure and cell type specificity of expression.Methods to isolate a receptor are made available by the ability to makepurified 499E9 for screening programs. Soluble or other constructs usingthe 499E9 sequences provided herein will allow for screening orisolation of 499E9 specific receptors. Many methods exist for expressioncloning, panning, affinity isolation, or other means to identify areceptor.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

EXAMPLES General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed.), vols. 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene and Wiley, New York; Innis, etal. (eds.) (1990) PCR Protocols: A Guide to Methods and Applications,Academic Press, N.Y. Methods for protein purification include suchmethods as ammonium sulfate precipitation, column chromatography,electrophoresis, centrifugation, crystallization, and others. See, e.g.,Ausubel, et al. (1987 and periodic supplements); Deutscher (1990) “Guideto Protein Purification” in Methods in Enzymology vol. 182, and othervolumes in this series; and manufacturer's literature on use of proteinpurification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad,Richmond, Calif. Combination with recombinant techniques allow fusion toappropriate segments, e.g., to a FLAG sequence or an equivalent whichcan be fused via a protease-removable sequence. See, e.g., Hochuli(1989) Chemische Industrie 12:69-70; Hochuli (1990) “Purification ofRecombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;and Crowe, et al. (1992) QIAexpress: The High Level Expression & ProteinPurification System QUIAGEN, Inc., Chatsworth, Calif. Cell culturetechniques are described in Doyle, et al. (eds.) (1994) Cell and TissueCulture: Laboratory Procedures, John Wiley and Sons, NY.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofPlow Cytometry Methods Wiley-Liss, New York, N.Y. Fluorescent labelingof appropriate reagents was performed by standard methods.

Example 1 Cloning of Mouse 499E9

Production of 3W Th1 or Th2 cells is described in Openshaw, et al.(1995) J. Exp. Med. 182:1357-1367. Briefly, Th1 or Th2 populations werederived from CD4+ T cells stimulated with antigen and antigen presentingcells in the presence of IL-12 or IL-4. Cells were stimulated once eachweek for 3 weeks, then harvested and restimulated, e.g., with PMA andionomycin for 4 h. See Murphy, et al. (1996) J. Exp. Med. 183:901-913.

Total RNA can be isolated, e.g., using the guanidine thiocyanate/CsClgradient procedure as described by Chirgwin, et al. (1978) Biochem.18:5294-5299. Poly(A)+RNA is isolated using, e.g., the OLIGOTEX mRNAisolation kit (QIAGEN). Such RNA from these cells is used to synthesizefirst strand cDNA, e.g., by using NotI/Oligo-dT primer (Gibco-BRL,Gaithersburg, Md.). Double-stranded cDNA is synthesized, ligated withBstXI adaptors, digested with NotI, size fractionated for >0.5 kilobasepairs (kb) and ligated into the NotI/BstXI sites of pJFE-14, aderivative of the pCDSRα vector. See Takebe, et al. (1985) Mol. Cell.Biol. 8:466-472. Electro-competent E. coli DH10α cells (Gibco-BRL) areused for transformation.

Independent clones were randomly picked and screened by hybridizationusing a cocktail of known cytokine cDNA's. Plasmid DNA's were preparedfrom clones that did not hybridize to the cytokine probes. These cloneswere grouped by insert size and further characterized by DNA sequencing.Clones corresponding to the 499E9 were isolated.

Example 2 Cellular Expression of Mouse 499E9

A probe specific for cDNA encoding mouse 499E9 is used to determinetissue distribution of message encoding the antigen. Standardhybridization probes may be used to do a Northern analysis of RNA fromappropriate sources, either cells, e.g., stimulated or in variousphysiological states, in various tissues, e.g., spleen, liver, thymus,lung, etc., or in various species. Southern analysis of cDNA librariesmay also provide valuable distribution information. Standard tissueblots or species blots are commercially available. Similar techniqueswill be useful for evaluating diagnostic or medical conditions which maycorrelate with expression in various cell types.

PCR analysis using appropriate primers may also be used. Antibodyanalysis, including immunohistochemistry or FACS, may be used todetermine cellular or tissue distribution.

Example 3 Purification of 499E9 Protein

Multiple transfected cell lines are screened for one which expresses theantigen, membrane bound or soluble forms, at a high level compared withother cells. Various cell lines are screened and selected for theirfavorable properties in handling. Natural 499E9 can be isolated fromnatural sources, or by expression from a transformed cell using anappropriate expression vector. Purification of the expressed protein isachieved by standard procedures, or may be combined with engineeredmeans for effective purification at high efficiency from cell lysates orsupernatants. FLAG or Hiss segments can be used for such purificationfeatures.

Example 4 Isolation of Homologous 499E9 Genes

The 499E9 cDNA can be used as a hybridization probe to screen a libraryfrom a desired source, e.g., a primate cell cDNA library. Many differentspecies can be screened both for stringency necessary for easyhybridization, and for presence using a probe. Appropriate hybridizationconditions will be used to select for clones exhibiting specificity ofcross hybridization.

Screening by hybridization or PCR using degenerate probes based upon thepeptide sequences will also allow isolation of appropriate clones.Alternatively, use of appropriate primers for PCR screening will yieldenrichment of appropriate nucleic acid clones.

Similar methods are applicable to isolate either species, polymorphic,or allelic variants. Species variants are isolated using cross-specieshybridization techniques based upon isolation of a full length isolateor fragment from one species as a probe.

Alternatively, antibodies raised against mouse 499E9 will be used toscreen for cells which express cross-reactive proteins from anappropriate, e.g., cDNA library. The purified protein or definedpeptides are useful for generating antibodies by standard methods, asdescribed above. Synthetic peptides or purified protein are presented toan immune system to generate monoclonal or polyclonal antibodies. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; andHarlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. The resulting antibodies are used, e.g., for screening,panning, or sorting.

Example 5 Preparation of Antibodies Specific for 499E9

Synthetic peptides or purified protein are presented to an immune systemto generate monoclonal or polyclonal antibodies. See, e.g., Coligan(1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press.Polyclonal serum, or hybridomas may be prepared. In appropriatesituations, the binding reagent is either labeled as described above,e.g., fluorescence or otherwise, or immobilized to a substrate forpanning method.

Example 6 Isolation of a Receptor for 499E9

A 499E9 construct expression product can be used as a specific bindingreagent to identify its binding partner, e.g., receptor, by takingadvantage of its specificity of binding, much like an antibody would beused. A 499E9 reagent is either labeled as described above, e.g.,fluorescence or otherwise, or immobilized to a substrate for panningmethods.

The binding composition is used to screen an expression library madefrom a cell line which expresses a binding partner, i.e. receptor.Standard staining techniques are used to detect or sort intracellular orsurface expressed receptor, or surface expressing transformed cells arescreened by panning. Screening of intracellular expression is performedby various staining or immunofluorescence procedures. See also McMahan,et al. (1991) EMBO J. 10:2821-2832.

Alternatively, 499E9 reagents are used to affinity purify or sort outcells expressing a receptor. See, e.g., Sambrook, et al. or Ausubel, etal.

Another strategy is to screen for a membrane bound receptor by panning,The cDNA containing receptor cDNA is constructed as described above. Theligand can be immobilized and used to immobilize expressing cells.Immobilization may be achieved by use of appropriate antibodies whichrecognize, e.g., a FLAG sequence of a 499E9 fusion construct, or by useof antibodies raised against the first antibodies. Recursive cycles ofselection and amplification lead to enrichment of appropriate clones andeventual isolation of receptor expressing clones.

Phage expression libraries can be screened by 499E9. Appropriate labeltechniques, e.g., anti-FLAG antibodies, will allow specific labeling ofappropriate clones.

Further studies have been performed by others, which support the scopeand breadth of the present disclosure and invention. Two reports haveappeared since the date of Applicants' priority filing. See, e.g., Wong,et al. (1997) J. Biol. Chem. 272:2510-25194; and Anderson, et al. (1997)Nature 390:175-179. These reports verify that other mammaliancounterparts do exist, and that a receptor for the ligand also exists.Moreover, they establish significant biology for the genes, asdescribed.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1-41. (canceled)
 42. A monoclonal antibody that specifically binds apolypeptide having 100% sequence identity with at least 12 contiguousresidues of SEQ ID NO:
 2. 43. The monoclonal antibody of claim 42wherein said polypeptide consists of a polypeptide having at least 90%sequence identity to residues 205 to 316 of SEQ ID NO:
 2. 44. Themonoclonal antibody of claim 42 wherein said polypeptide is soluble. 45.The antibody of claim 42, wherein the antibody is recombinantlyproduced.
 46. The antibody of claim 45, wherein the antibody is achimeric, humanized, or human antibody.
 47. A monoclonal antibody thatspecifically binds a polypeptide having 100% sequence identity with atleast 16 contiguous residues of SEQ ID NO:
 2. 48. The monoclonalantibody of claim 47 wherein said polypeptide consists of a polypeptidehaving at least 90% sequence identity to residues 205 to 316 of SEQ IDNO:
 2. 49. The monoclonal antibody of claim 47 wherein said polypeptideis soluble.
 50. The antibody of claim 47, wherein the antibody isrecombinantly produced.
 51. The antibody of claim 50, wherein theantibody is a chimeric, humanized, or human antibody.
 52. A monoclonalantibody that specifically binds a polypeptide having 100% sequenceidentity with at least 20 contiguous residues of SEQ ID NO:
 2. 53. Themonoclonal antibody of claim 52 wherein said polypeptide consists of apolypeptide having at least 90% sequence identity to residues 205 to 316of SEQ ID NO:
 2. 54. The monoclonal antibody of claim 52 wherein saidpolypeptide is soluble.
 55. The antibody of claim 52, wherein theantibody is recombinantly produced.
 56. The antibody of claim 55,wherein the antibody is a chimeric, humanized, or human antibody.
 57. Anantibody that specifically binds a polypeptide having at least about 90%sequence identity to residues 205 to 316 of SEQ ID NO: 2, wherein thepolypeptide comprises at least about 8 contiguous residues of SEQ ID NO:2 within residues 205 to 316 of SEQ ID NO:
 2. 58. An antibody thatspecifically binds a polypeptide having at least about 90% sequenceidentity to residues 205 to 316 of SEQ ID NO: 2, wherein the polypeptidecomprises at least about 12 contiguous residues of SEQ ID NO: 2 withinresidues 205 to 316 of SEQ ID NO:
 2. 59. An antibody that specificallybinds a polypeptide having at least about 90% sequence identity toresidues 205 to 316 of SEQ ID NO: 2, wherein the polypeptide comprisesat least about 16 contiguous residues of SEQ ID NO: 2 within residues205 to 316 of SEQ ID NO:2.
 60. An antibody that specifically binds apolypeptide having at least about 90% sequence identity to residues 205to 316 of SEQ ID NO: 2, wherein the polypeptide comprises at least about20 contiguous residues of SEQ ID NO: 2 within residues 205 to 316 of SEQID NO:
 2. 61. The antibody of claim 42, wherein the antibody binds a499E9 polypeptide, wherein binding of the antibody sterically blocksbinding of the 499E9 polypeptide to a binding partner of the 499E9polypeptide.
 62. The antibody of claim 61, wherein the binding partneris a ligand or receptor of the polypeptide.
 63. The antibody of claim57, wherein the antibody sterically blocks binding of a polypeptidehaving 90% sequence identity to residues 205 to 316 of SEQ ID NO: 2 to abinding partner of the 499E9 polypeptide.
 64. The antibody of claim 63,wherein the binding partner is a ligand or receptor of the polypeptide.65. A method of antagonizing the development of hematopoietic cellscomprising administering an antagonist of a 499E9 polypeptide, whereinthe antagonist comprises the antibody of claim
 42. 66. A method ofantagonizing the development of hematopoietic cells comprisingadministering an antagonist of a 499E9 polypeptide, wherein theantagonist comprises the antibody of claim
 57. 67. A method of treatinga T-cell mediated condition comprising administering a therapeuticallyeffective amount of an antibody of claim
 42. 68. A method of treating aT-cell mediated condition comprising administering a therapeuticallyeffective amount of an antibody of claim
 57. 69. A method of modulatingT-cell mediated interactions with cells containing a receptor therefor,comprising administering a therapeutically effective amount of anantibody of claim 42, wherein the T-cell mediated interactions involveT-cells containing a 499E9 polypeptide.
 70. A method of modulatingT-cell mediated interactions with cells containing a receptor therefor,comprising administering a therapeutically effective amount of anantibody of claim 57, wherein the T-cell mediated interactions involveT-cells containing a 499E9 polypeptide.
 71. A method of treating acondition characterized by abnormal hematopoietic cell developmentcomprising administering a therapeutically effective amount of anantibody of claim
 42. 72. A method of treating a condition characterizedby abnormal hematopoietic cell development comprising administering atherapeutically effective amount of an antibody of claim 57.